Human immunodeficiency virus type 1 (HIV-1) is the cause of acquired immunodeficiency syndrome (AIDS), which is characterized by the depletion of CD4-positive lymphocytes (1,2). Infection of humans by HIV-1 typically involves an initial period of acute, high-level viremia, followed by a chronic, low-level viremia (3-6). It is thought that the antiviral immune response helps to determine the “set-point” for chronic viremia. HIV-1 persistence results in progressive CD4-positive lymphocyte decline, which ultimately compromises the immune response, including that directed against HIV-1(6). The resulting resurgence of high-level viremia is a harbinger of poor clinical outcome (6,7).
The envelope protein of a lentivirus is the most visible portion of the virion because it is on the surface of the virus particle. Thus, considerable attention has focussed on the envelope protein as a target for inhibiting viral entry. Strategies that have been used include using the envelope protein to generate an immune response, decoys for the envelope protein, etc. These approaches have not yet been successful.
It was recently reported that a large scale clinical trial was going to be attempted with an HIV envelope protein as an immunogen. While the initial trials with the protein have not been reported to be promising in terms of showing any significant protective immunity, they have also not indicated any significant harm caused by the vaccine candidate. The fact that a clinical trial with this type of preliminary results would be attempted shows the importance placed upon the use of the envelope protein and the need for improvements in the envelope protein that can be used such as by selectively enhancing its immunogenicity.
HIV-1 is tropic for CD4-positive cells by virtue of the high affinity interaction between the HIV-1 exterior envelope glycoprotein, gp 120, and CD4, which acts as the primary virus receptor (1,2,3). The gp 120 molecule is derived by proteolytic cleavage from the precursor gp 160 glycoprotein and noncovalently associates with the transmembrane glycoprotein, gp41, to form trimeric complexes on the virus or cell surface (4,5,6,7). The gp 160 precursor is initially modified by the addition of high-mannose sugars at approximately 28 N-linked glycosylation sites of the approximately 845-870 amino acid primary translation product. A subset of the high-mannose sugars becomes modified to complex carbohydrates following oligomerization and cleavage of the gp 160 precursor in the Golgi apparatus.
The envelope protein is an attractive target because, like that of other retroviruses, the entry of HIV-1 into target cells is mediated by the viral envelope glycoproteins, gp 120 and gp41, which are derived from a gp 160 precursor (See, Allan, J S, et al., “Major Glycoprotein Antigens That Induce Antibodies in AIDS Patients are Encoded by HTLV-III,” Science 228:1091-1093 (1985); Robey, W G., et al., “Characterization of Envelope and Core Structural Gene Products of HTLV-III with Sera from AIDS Patients,” Science 228:593-595 (1985)). The gp 160 glycoprotein is created by the addition of N-linked, high mannose sugar chains to the approximately 845-870 amino acid primary translation product of the env gene in the rough endoplasmic reticulum. Trimerization of gp 160 in the endoplasmic reticulum is mediated by the formation of a coiled coil within the gp41 ectodomain. (See, Earl, P L., et al., “Oligomeric Structure of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein,” Proc. Natl. Acad. Sci. USA 87:648-652 (1990); Pinter, A., et al., “Oligomeric Structure of gp41, the Transmembrane Protein of Human Immunodeficiency Virus Type 1, ” J. Virol. 63:2674-2679; Lu, M., et al., “A Trimeric Structural Domain of the HIV-1 Transmembrane Glycoprotein,” Nature Structural Biol. 2:1075-1082 (1995); Chan, D C, et al., “Core Structure of gp41 from the HIV Envelope Glycoprotein,” Cell 89:263-273 (1997); and Weissenhorn, W., et al., “Atomic Structure of the Ectodomain from HIV-1 gp41,” Nature 387:426-430 (1997)). The gp 160 trimers are transported to the Golgi apparatus, where cleavage by a cellular protease generates the mature gp 120 and gp41 glycoproteins, which remain associated through non-covalent interactions (Earl, P L, et al., “Folding, Interaction with GRP78-BiP, Assembly and Transport of the Human Immunodeficiency Virus Type 1 Envelope Protein,” J. Virol. 65:2047-2055 (1991); and Kowalski, M., et al., “Functional Regions of the Envelope Glycoprotein of Human Immunodeficiency Virus Type 1,” Science 237:1351-1355 (1987)). In mammalian host cells, addition of complex sugars to selected, probably surface-exposed, carbohydrate side chains of the envelope glycoproteins occurs in the Golgi apparatus. (See, Leonard, C K, et al., “Assignment of Intra chain Disulfide Bonds and Characterization of Potential glycosylation Sites of the Type 1 Recombinant Human Immunodeficiency Virus Envelope Glycoprotein (gp 120) Expressed in Chinese Hamster Ovary Cells,” J. Biol. Chem. 265:10373-10382 (19990)).
Most of the surface-exposed elements of the oligomeric envelope glycoprotein complex are contained on the gp 120 exterior envelope glycoprotein. (See, Moore, J., et al., “Probing the Structure of the Human Immunodeficiency Virus Surface Glycoprotein gp 120 with a Panel of Monoclonal Antibodies,” J. Virol. 68:469-484 (1994)). When the gp 120 glycoproteins derived from different primate immunodeficiency viruses are compared, five conserved regions (C1 to C5) and five variable regions (V1 to V5) can be identified. (See, Starcich, B R, et al., “Identification and Characterization of Conserved and Variable Regions of the Envelope Gene HTLV-III/LAV, the Retrovirus of AIDS,” Cell 45:637-648 (1986); Myers, G., et al. “Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences,” Los Alamos National Laboratory, (1994)). Intramolecular disulfide bonds in the gp 120 glycoprotein result in the incorporation of the first four variable regions into large, loop-like structures. Antibody binding studies and deletion mutagenesis have indicated that the major variable loops are well-exposed on the surface of the gp 120 glycoprotein. (See, Wyatt, R., et al., “Functional and Immunologic Characterization of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Containing Deletions of the Major Variable Regions,” J. Virol. 7:4557-4565 (1993); Pollard, S., et al., “Truncated Variants of gp 120 bind CD4 with High Affinity and Suggest a Minimum CD4 Binding Region,” EMBO J. 11:585-591 (1992)).
The mature envelope glycoprotein complex is incorporated into HIV-1 virions, where it mediates virus entry into the host cell. The gp 120 exterior glycoprotein binds the CD4 glycoprotein, which serves as the primary receptor. (See, Klatzmann, D., et al., “T-lymphocyte T4 Molecule Behaves as the Receptor for Human Retrovirus LAV,” Nature London 312:767-768 (1984); and Dalgleish, A G., et al., “The CD4 (T4) Antigen is an Essential Component of the Receptor for the AIDS Retrovirus,” Nature 312:763-767 (1984)). The association of gp 120 with CD4 is mediated by the interaction of a discontinuous gp 120 structure with the CDR2-like region of the CD4 amino-terminal domain. (See, Brodsky, M H., et al., “Analysis of the Site in CD4 that Binds to the HIV Envelope Glycoprotein,” J. Immunol. 144:3078-3086 (1990); Peterson, A., et al., “Genetic analysis of Monoclonal Antibody and HIV binding Sites on the Human Lymphocyte Antigen CD4,” Cell 54:65-72 (1988); Moebius, U., et al., “The Human Immunodeficiency Virus gp 120 Binding Site on CD4: Delineation by quantitative Equilibrium and Kinetic Binding Studies of Mutants in Conjunction with a High-Resolution CD4 Atomic Structure,” J. Exp. Med. 176:507-517 (1982); Arthos, J., et al., “Identification of the Residues in Human CD4 Critical for the binding of HIV,” Cell 57:469 (1989); Ryu S E., et al., “Crystal Structure of an HIV-binding Recombinant Fragment of Human CD4,” Nature London 348:419-425 (1990); and Wang, J., et al., “Atomic Structure, of a Fragment of Human CD4 containing Two immunoglobulin-like Domains,” Nature London 348:411-418 (1990)). Amino acids in the gp 120 C3 and C4 regions have been implicated in CD4 binding. (See, Cordonnier, A., et al., “Single Amino Acid Changes in HIV Envelope Affect Viral Tropism and Receptor Binding, Nature 340:571-574 (1989); Lasky, L., et al., “Delineation of a Region of the Human Immunodeficiency Virus Type 1 gp 120 Glycoprotein Critical for Interaction with the CD4 Receptor,” Cell 50:975-985 (1987); and Olshevsky, U., et al., “Identification of Individual HIV-1 gp 120 Amino Acids Important for CD4 Receptor Binding,” J. Virol. 64:5701-5707 (1990)). The association of gp 120 with CD4 is believed to initiate conformational changes in the HIV-1 envelope glycoprotein complex, leading to interactions with members of the chemokine receptor family. (See, Sattentau, Q., et al., “Conformational Changes Induced in the Human Immunodeficiency Virus Envelope Glycoprotein by Soluble CD4 binding,” J. Exp. Med. 174:407-415 (1991); Thali, M., et al., “Characterization of Conserved Human Immunodeficiency Virus Type 1 (HIV-1) gp 120 neutralization Epitopes Exposed Upon gp 120-CD4 Binding,” J. Virol 67:3978-3988 (1993); Sattentau, Q., et al., “Conformational Changes Induced in the Envelope Glycoproteins of Human and Simian Immunodeficiency Virus by Soluble Receptor Binding,” J. Virol. 67:7388-7393 (1993); Trkola, A., et al., “CD4-dependent, antibody-sensitive Interactions Between HIV-1 and its Co-receptor CCRO5,” Nature 384:184-187 (1996); and WU, L., et al., “CD4-induced Interaction of Primary HIV-1 gp 120 Glycoproteins with the Chemokine Receptor CCR5,” Nature 384:179-183 (1996).
Chemokine receptors are G protein-coupled, seven-membrane-spanning proteins involved in leukocyte chemotaxis. (See, Baggioline, M., et al., “Interleukin-8 and Related Chemotactic Cytokines-CXC and CC Chemokines,” Adv. Immunol. 55:97-179 (1994); Gerard, N., et al., “the Pro-Inflammatory Seven-Transmembrane-Segment Receptors of the Leukocyte,” Curr. Opin. Immunol. 6:140-145 (1994); and Premack, B A., et al, “Chemokine Receptors: Gateways to Inflammation and Infection,” Nature Medicine 11:1174-1178 (1996)). Most laboratory-adapted HIV-1 viruses utilize a CXC chemokine receptor called CXCR4 (also called LESTR, HUMSTSR or fusin), while most macrophage-tropic primary HIV-1 viruses use the CC chemokine receptor CCR5 (see, Feng, Y., et al., Science 272:872-877 (1996); Choe, H., et al., Cell 85:1135-1148 (1996); Deng, H K., et al., Nature 381:661-666 (1996); Dragic, T., et al., Nature 381:667-673 (1996); Doranz, B J., et al., Cell 85:1149-1158 (1996); and Alkhatib, G., et al., Science 272:1955-1958 (1996)), and to an extent CCR3 or CCR2. Primary dual-tropic HIV-1 isolates use CCR5 as well as CXCR4. (See, Zhang, L., et al., Nature 383:768 (1996) and Connor, R., et al., J. Exp. Med. 185:21-628 (1997)). The macrophage-tropic primary viruses are those most often transmitted from infected to uninfected individuals, and predominate during the long, asymptomatic phase of infection. (See, Cheng-Mayer, C., et al., Science 240:80-82; Zhu, T., et al., Science 261:1179-1181 (1993); Fenyo, E., J. Virol. 62:4414-4419 (1988); Schuitemaker, H., et al., J. Virol. 66:1354-1360 (1991); and Connor, R I., et al., J. Virol. 67:1772-1778 (1993)). The importance of CCR5 for HIV-1 transmission is underscored by the observation that humans with homozygous defects in CCR5 are relatively resistant to HIV-1 infection. (See, Liu, R., et al., Cell 86:367-378 (1996); Samson, M., et al., Nature 10 382:722-725 (1996); and Dean M., et al., Science 273:1856-1862 (1996)). CCR5 is used as a corrector by almost all primary HIV-1 isolates regardless of geographic clade, and is used by the related human and primate immunodeficiency viruses, HIV-2 and simian immunodeficiency virus, SIV. (See, Marcon, L., et al., J. Virol 71:2522-2527 (1997); Chen, Z., et al., J Virol. 71:2705-2714 (1997); and Cocchi, F., et al., Science 270:1811-1815 (1995)). This suggests that at least part of the viral binding site for CCR5 is well-conserved among these immunodeficiency viruses. While these gp 120 structures are under investigation and have yet to be completely defined, mutagenic studies have suggested that elements of the V3 loop may constitute part of the chemokine receptor binding site. Genetic studies of viruses with chimeric HIV-1 envelope glycoproteins containing different V3 loops demonstrated that the gp 120 V3 region is a major determinant of which chemokine receptor, CCRS or CXCR4, can be used as an,entry cofactor. (See, Cocchi, F., et al., Nature med., 2:1244-1247 (1996); and Speck, R., et al., J. Virol. (in press)). Thus, even in the relatively variable background of the V3 domain, there may exist conserved structural features that collaborate with other conserved gp 120 structures to create a high-affinity binding site for CCR5.
It is likely that the interaction of the gp 120-CD4 complex with the appropriate chemokine receptor promotes additional conformational changes in the envelope glycoprotein complex. By analogy with the influenza hemoglutinin, it has been suggested that the HIV-1 gp41 ectodomain undergoes major conformational changes during virus entry. (See, Carr, C M., et al., Cell 73:823-832 (1993); Chen, C H., et al., J. Virol. 69:3771-3777 (1995); Bullough, P., et al. Nature 371:37-43 (1994); and Weissenhorn, W., et al., EMBO J 15:1507-1514 (1996)). The proposed result of these changes is the insertion of the hydrophobic gp41 amino terminus (the “fusion peptide”) into the membrane of the target cell. Mutagenic analysis and the recently determined crystal structures of HIV-1 gp41 ectodomain fragments are consistent with this model (see, Freed, E., et al., Proc. Natl. Acad. Sci USA 87:4650-4654 (1990)).
The exposed nature of the HIV-1 envelope glycoproteins on the surface of virions or infected cells renders them prime targets for the antiviral immune response. In fact, the only viral proteins accessible to neutralizing antibodies are the envelope glycoproteins. Neutralizing antibodies appear to be an important component of a protective immune response, in chimpanzees challenged with HIV-1 (see, Berman, P W., et al., Nature 345:622-625 (1990); Girard, et al., Proc. Natl. Acad. Sci. USA 88:542-546 (1991); Emini, et al., Nature 355:728-730 (1991); and Bruck, et al., Vaccine 12:1141-1148 (1994). That neutralizing antibodies generated during the course of HIV-1 infection do not provide permanent antiviral effect may in part be due to the generation of neutralization escape virus variants (see, Nara, et al., J. Virol. 64:3779-3791 (1990); Gegerfelt, et al., Virology 185:162-168 (1991); and Arendrup, et al., J AIDS 5:303-307 (1992)), and to the general decline in the host immune system associated with pathogenesis.
HIV-1 neutralizing antibodies are mostly directed against linear or discontinuous epitopes of the gp 120 exterior envelope glycoprotein. Rare examples of gp41-directed neutralizing antibodies have also been documented (see, Muster, et al., J. Virol. 67:6642-6647 (1993)). Neutralizing antibodies that arise early in infected humans and that are readily generated in animals by immunization are primarily directed against linear neutralizing determinants in the third variable (V3) loop of gp 120 glycoprotein (see, Matthews, et al., Proc. Natl. Acad. Sci. USA 83:9709-9713 (1986); and Javaherian, et al., Science 250:1590-1593 (1990)). These antibodies generally exhibit the ability to neutralize only a limited number of HIV-1 strains, although some subsets of anti-V3 antibodies recognize less variable elements of the region and therefore exhibit broader neutralizing activity (see, Ohno, et al., Proc. Natl. Acad. Sci. USA 88:10726-10729 (1991); Moore, et al., J. Virol. 69:122-133 (1995); and Gorny, et al., J. Virol. 66:7538-7542 (1992)). Envelope glycoprotein variation within the linear V3 epitope and outside of the epitope can allow escape of viruses from neutralization by these antibodies (see, McKeating, et al., J. Virol. 67:4932-4944 (1993)). The second variable (V2) region of the HIV-1 envelope glycoprotein has also been shown to be a target for strain-restricted neutralizing antibodies (see, Fung, et al., J. Virol. 66:848-856 (19921; Moore, et al., J. Virol. 67:6136-6151 (1993)). Most of the V2 epitopes consist of continuous but conformation-dependent determinants.
Later in the course of HIV-1 infection of humans, antibodies capable of neutralizing a wider range of HIV-1 isolates appear (see, Profy, et al., J. Immunol. 144:4641-4647 (1990); Berkower, et al., J. Exp. Med. 170: 1681-1695 (1989); Ho, et al., J. Virol. 489-493 (1991); Kang, et al., Proc. natl. Acad. Sci. USA 88:6171-6175 (1991); Steimer, et al., Science 254:105-108 ((1991); and Moore et al., J. Virol. 67:863-875 (1993)). These broadly-neutralizing antibodies have been difficult to elicit in animals (see, Rusche et al., Proc. Natl. Acad. Sci. USA 84:6924-6928 (1987); Klaniecki et al., AIDS Res. Hun; Retro. 7:791-798 (1991); and Haigwood, et al., J. Virol. 66:172-182 (1992)), and are not merely the result of additive anti-V3 loop reactivities against diverse HIV-1 isolates that accumulate during active infection. A subset of the broadly reactive antibodies, found in most HIV-1-infected individuals, interferes with the binding of gp 120 and CD4. At least some of these antibodies recognize discontinuous gp 120 epitopes (the so-called CD4BS epitopes) present only on the native glycoprotein. Human monoclonal antibodies derived from HIV-1-infected individuals have been identified that recognize the gp 120 glycoproteins from a diverse range of HIV-1 isolates, that block gp 120-CD4 binding, and that neutralize virus infection (see, Posner, et al., J. Immunol. 146:4325-4332 (1991); and Tilley, et al., Res. Virol. 142:247-259 (1991)). Some of these CD4BS-directed antibodies efficiently neutralize primary HIV-1 isolates (see, Burton, et al., Science 266:1024-1027 (1994)), which are generally more resistant to neutralization than are viruses passaged in immortalized cell lines (see, Daar, et al., Proc. Natl. Acad. Sci. USA 87:6574-6578 (1990); Wrin, et al., J. virol.69:39-48 (1995); Sullivan, et al., J. Virol. 69:4413-4422 (1995); Sawyer, et al., J. Virol. 67:1342-1349 (1994); Moore, et al., J. Virol. 69:101-109 (1995); and D'Souza, et al., J. Infect. Dis. 175:(in press)(1997)). The discontinuous epitopes recognized by many of the human monoclonal antibodies directed against the CD4BS epitopes have been characterized by mutagenic analysis (see, Thali, et al., J. Virol. 65:6188-6193 (1991); Thali, et al., J. Virol. 66:5635-5641 (1992); McKeating, et al., Virology 190:134-142 (1992)). Amino acid changes in seven discontinuous gp 120 regions, four of which overlap regions defined to be important for CD4 binding, disrupt recognition by these antibodies and, in some cases, allow the generation of neutralization escape mutants.
A second group of neutralizing antibodies found in a smaller number of HIV-1-infected humans is directed against conserved gp 120 epitopes that are exposed better upon CD4 binding (see, Thali, et al., J. Virol. 67:3978-3988 (1993)). These epitopes, referred to as the CD4-induced (CD4i) epitopes, are extremely sensitive to conformational changes in the gp 120 glycoprotein. The integrity of these epitopes is affected by gp 120 amino acid changes in the conserved stem of the V1/V2 stem-loop structure and in the C4 region. The CD4i epitopes have been shown to be proximal to the V3 loop and to be masked by the V1/V2 variable loops (see, Wyatt, et al., J. Virol. 69:5723-5733 (1995); and Moore, et al., J. Virol 70:1863-1872 (1996)). It has been shown that CD4 binding induces a movement of the V1/V2 loops that exposes the CD4i epitopes. Interestingly, it has been shown that neutralizing antibodies directed against either the V3 loop or the CD4i epitopes block the ability of gp 120-CD4 complexes to bind CCR5. Thus, it appears that the major groups of neutralizing antibodies generated in HIV-1-infected humans block the binding of virus to its cellular receptors, either CD4 or the chemokine receptors.
The development of an HIV-1 vaccine as explained above has been hampered by the inefficiency with which antibodies directed against the more conserved gp 120 structures are elicited. Most of the antibodies elicited by the HIV-1 envelope glycoproteins, either in infected humans or chimps or in animals immunized with envelope glycoprotein preparations, are not able to neutralize virus. Many of these non-neutralizing antibodies are directed against gp 120 structures that are inaccessible on the native envelope glycoprotein complex due to interaction with the gp41 ectodomain (see, Wyatt, et al., (1997)). When neutralizing antibodies are elicited, these are often directed against variable portions of the HIV-1 envelope glycoproteins. Most of the neutralizing antibodies elicited by native HIV-1 gp 120 or gp 160 glycoproteins are directed against the V3 loop (see, Haigwood, et al., AIDS Res. Hum. Retro. 6:855-869 (1990)). Multiple immunizations with native gp 120 or gp 160 glycoproteins are required to elicit even low titers of neutralizing antibodies with broader strain reactivity. This same pattern of elicitation of neutralizing antibodies has been observed in HIV-1-infected humans or chimps, with antibodies directed against the V3 loop appearing earlier in infection. These results suggest that the structure of the HIV-1 gp 120 envelope glycoprotein has evolved to decrease the immunogenicity of particular epitopes in which variation is poorly tolerated by the virus. By the time immune responses to these epitopes are elicited, immune compromise has occurred, viral burden is high, and virus variation and the potential for neutralization escape has reached significant levels. These considerations suggest that use of the native, complete HIV-1 glycoprotein as an immunogen will most efficiently elicit the same types of immune responses that the virus has evolved to evade most efficiently. Improved immunogens based upon the envelope protein are necessary.
Previous studies have indicated that the relatively poor surface accessibility of the more conserved gp 120 epitopes related to the CD4 and chemokine receptor binding sites may in part provide an explanation for the low apparent immunogenicity of these regions (21). The V1/V2 and V3 variable loops of the HIV-1 gp 120 glycoprotein have been shown to mask the CD4BS epitopes, and removal of these variable regions results in a 5-50-fold increase in exposure of most of the CD4BS epitopes, on both the monomeric and the multimeric envelope glycoproteins (21). Removal of the V1 and V2 variable loops results in an increased exposure of HIV-1 gp 120 epitopes (V3 and CD4i epitopes) located near the binding site for the chemokine receptors. Thus, both of the receptor-binding regions of the HIV-1 gp 120 glycoprotein are partially masked by the large variable loop structures of the glycoprotein.
It is imperative that means of efficiently eliciting an array of antibodies directed against the more conserved gp 120 elements be developed.